1. Field of the Invention
The present invention relates to a process for producing distilled liquors having an excellent flavor by the use of yeast into which a ferulic acid decarboxylase gene has been introduced.
Ferulic acid decarboxylase is an enzyme which catalyzes the decarboxylation of ferulic acid to form 4-vinylguaiacol.
2. Brief Description of the Background Art
In the liquor industry, it is always desired to develop distilled liquors such as shochu (a Japanese distilled liquor), bai jiu (a Chinese distilled liquor), whiskey, brandy, vodka, rum, gin and the like having an excellent flavor. Distilled liquors possesing an excellent flavor typically have a relatively high content of at least one of 4-vinylguaiacol, vanillin, or vanillic acid.
Vanillin and vanillic acid are formed by oxidation of 4-vinylguaiacol [Nippon Nogeikagaku Kaishi, 70(6), 684-686 (1996)].
It is known that distilled liquors having an excellent flavor can be produced by adding hydroxycinnamic acid ester hydrolase, or a koji mold having a high productivity of hydroxycinnamic acid ester hydrolase, (Japanese Published Unexamined Patent Application No. 115957/95) or ferulic acid esterase [Nippon Nogeikagaku Kaishi, 70(6), 684-686 (1996)] to liberate ferulic acid into moromi.
Ferulic acid, as well as cinnamic acid and coumaric acid, is a kind of phenylacrylic acid, and is bonded in the form of ester to the arabinose side chain of arabinoxylan contained in the hemicellulose fraction which constitutes cell walls of plants such as cereals.
A method for preparing yeast having a high ferulic acid decarboxylase activity by cell fusion has been proposed [Abstracts of the Annual Meeting of the Society of Fermentation and Bioengineering, 41 (1995)]. However, it has not yet been possible to obtain yeast having an increased ferulic acid decarboxylase activity. Accordingly, a method of producing distilled liquors having an excellent flavor by the use of such yeast is not known.
Ferulic acid decarboxylase of Bacillus pumilus [Appl. Environ. Microbiol., 61(1), 326-332 (1995)] and that of Pseudomonas fluorescens [J. Bacteriol., 176, 5912-5918 (1994)] have already been isolated and purified. The gene encoding ferulic acid decarboxylase (hereinafter referred to as FDC gene) of B. pumilus [Appl. Environ. Microbiol., 61, 4484-4486 (1995)] is also known. Ferulic acid decarboxylase of yeast belonging to the genus Saccharomyces, etc. has not been isolated, even though the existence of the enzyme activity has been recognized.
As to Saccharomyces cerevisiae, the gene encoding phenylacrylic acid decarboxylase (hereinafter referred to as PAD1 gene) [Gene, 142, 107-112 (1994)] is known, but there is no report on its FDC gene.
An object of the present invention is to provide ferulic acid decarboxylase which is useful in the production of distilled liquors having an excellent flavor.
The present invention relates to a protein having the amino acid sequence represented by SEQ ID NO: 1, or a protein having ferulic acid decarboxylase activity and having an amino acid sequence wherein one or more amino acid residues are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1 (hereinafter referred to as the protein of the present invention); a gene encoding said protein (hereinafter referred to as the gene of the present invention); a recombinant vector comprising said gene (hereinafter referred to as the recombinant vector of the present invention); a transformant carrying said recombinant vector (hereinafter referred to as the transformant of the present invention); a process for producing 4-vinylguaiacol, vanillin, or vanillic acid, which comprises bringing ferulic acid into contact with an enzyme source having ferulic acid decarboxylase activity which is derived from said transformant in an aqueous medium to form 4-vinylguaiacol, vanillin, or vanillic acid in the aqueous medium, and recovering 4-vinylguaiacol, vanillin, or vanillic acid therefrom (hereinafter referred to as the process for producing 4-vinylguaiacol, vanillin, or vanillic acid of the present invention); a process for producing a distilled liquor, which comprises adding an enzyme source having ferulic acid decarboxylase activity which is derived from said transformant to moromi (hereinafter referred to as the process for producing a distilled liquor by using the protein of the present invention); and a process for producing a distilled liquor, which is characterized in that yeast having an enhanced ferulic acid decarboxylase activity is used (hereinafter referred to as the process for producing a distilled liquor by using the yeast of the present invention).
The present invention provides a distilled liquor produced by the above-mentioned processes for producing a distilled liquor.